ABB
Archives of Biochemistry and Biophysics 409 (2003) 172–179
www.elsevier.com/locate/yabbi
Evaluation of thyroid hormone effects
on liver P450 reductase translation
Ekaterina V. Apletalina, Huan-Chen Li, and David J. Waxman*
Department of Biology, Boston University, 5 Cummington Street, Boston, MA 02215, USA
Received 30 May 2002, and in revised form 19 July 2002
Abstract
The expression of NADPH cytochrome P450 oxidoreductase (P450R) in rat liver is positively regulated by thyroid hormone (T3),
at both the transcriptional and post-transcriptional levels. Here we investigate the effects of T3-induced hyperthyroidism on the
regulation of P450R protein synthesis. T3 treatment of adult male rats led to a strong induction (up to 10-fold) of liver P450R
mRNA but little or no change in P450R protein and activity. Investigation of this discrepancy revealed that the association of
hepatic P450R mRNA with polysomes was not altered by T3 treatment, suggesting that the discoordinate changes in P450R mRNA
and protein levels do not reflect decreased recruitment of T3-induced P450R mRNA into polysomes. Moreover, polysome size
distribution analysis of P450R mRNA did not show any T3-dependent changes. When assayed in an in vitro translation system, T3induced and uninduced P450R mRNAs were translated with similar efficiencies. Moreover, liver cell extract from T3-treated rats did
not selectively inhibit in vitro translation of T3-induced P450R mRNA. Thus, neither structural changes in P450R mRNA nor transacting binding proteins in liver cytosol were found to control translation of P450R mRNA in response to T3 treatment. Taken
together, these data suggest that P450R may in part be regulated at the level of protein stability in hyperthyroid rat liver.
Ó 2002 Elsevier Science (USA). All rights reserved.
Keywords: Cytochrome P450 reductase; Thyroid hormone; Gene expression; Post-transcriptional regulation
NADPH cytochrome P450 oxidoreductase (P450R)1
is a ubiquitous flavoprotein that transfers electrons from
NADPH to several microsomal enzymes [1,2]. P450R is
best known for its role in transferring electrons to cytochrome P450 (P450), which catalyzes metabolism of a
wide range of lipophilic chemicals, including many
drugs, environmental carcinogens and endogenous steroids, fatty acids, and alkaloids [3–5]. P450R also provides electrons to microsomal heme oxygenase, which
oxidatively cleaves heme and plays an important role in
cellular heme homeostasis [6]. In addition to its involvement in P450- and heme oxygenase-dependent
*
Corresponding author. Fax: 1-617-353-7404.
E-mail address: djw@bu.edu .
1
Abbreviations used: P450R, NADPH-dependent cytochrome P450
oxidoreductase; P450, cytochrome P450; T3, 3,30 ,5-triiodo-L -thyronine, thyroid hormone; ON, oligonucleotide; PCR, polymerase chain
reaction; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel
electrophoresis; GST, glutathione-S-transferase; 50 - or 30 -UTR, 50 - or
30 -untranslated region.
metabolic processes, P450R catalyzes reductive activation of quinones, including several anticancer quinone
agents [7–9].
P450R is encoded by a single gene consisting of 16
exons [10]. P450R is most highly expressed in liver,
where it is subject to regulation by xenobiotics and by
endogenous hormones, including thyroid hormone (T3)
[1,11–14]. T3 regulates the expression of a variety of
proteins in mammalian tissues at different levels. Similarly to malic enzyme [15], apolipoprotein E [16], Na,KATPase alpha 3 subunit [17], and osteocalcin [18],
P450R is regulated by T3 at both the transcriptional and
post-transcriptional levels [12–14]. The transcriptional
activation of P450R by T3 is mediated by a thyroid
response element in the P450R promoter comprised of
three imperfect direct repeats of the thyroid response
motif AGGTCA [12]. T3 stimulates an increase in liver
P450R transcription, resulting in the induction of liver
nuclear P450R precursor RNA within 12 h of hormone
treatment [13]. The subsequent increase (up to 10-fold)
in cytoplasmic P450R mRNA [11] does not, however,
0003 9861/02/$ see front matter Ó 2002 Elsevier Science (USA) All rights reserved
E.V. Apletalina et al. / Archives of Biochemistry and Biophysics 409 (2003) 172–179
lead to increases in hepatic P450R protein or activity, a
finding that strongly suggests post-transcriptional regulation. T3 also induces a transient increase in P450R
mRNA nuclear polyadenylation as well as destabilization of cytoplasmic P450R mRNA [14]; however, the
consequences of these post-transcriptional effects of T3
are unknown.
The present study was undertaken to investigate the
post-transcriptional events that may underlie the discoordinate changes in P450R mRNA and P450R protein
and activity seen in hyperthyroid rat liver. We demonstrate that T3 does not alter the polyribosomal distribution of P450R mRNA. Moreover, using an in vitro
translation system, we show that there is no change in
the translational efficiency of P450R mRNA, either in
the presence or in the absence of T3-induced liver cell
extract. These findings are discussed in the context of
several potential mechanisms whereby liver P450R may
be regulated by changes in thyroid hormone status.
Materials and methods
Animals. Adult male Fischer 344 rats, housed under
standardized conditions of light and temperature, were
rendered hyperthyroid by a single i.p. injection of T3
(Sigma) at a receptor-saturating dose of 200 lg/100 g
body weight. Control animals received the same volume
(0.3–0.5 ml) of vehicle (0.01 M KPi , pH 9.5, containing
0.9% NaCl). Rats were killed 12–36 h later and individual livers stored frozen at )80 °C. Animal experiments included two or three animals per time point.
Where specified, total liver RNA and liver mRNA were
purified from livers of untreated and T3-treated hypothyroid rats. Induction of hypothyroidism using the
antithyroid drug methimazole (0.025% (w/v) in the
drinking water for 16 days) followed by T3 injection
(200 lg/100 g body weight) was described earlier [11].
RNA isolation. Total RNA was purified from frozen
liver samples using TRIzol reagent (Invitrogen) according to the manufacturerÕs instructions. mRNA was
purified from total RNA using Oligotex mRNA midi kit
(Qiagen).
Reverse transcription and real-time PCR. Total liver
RNA (0.4 lg) was reverse transcribed by murine leukemia virus reverse transcriptase (Applied Biosystems,
Foster City, CA) or OmniScript reverse transcriptase
(Qiagen) in a total reaction volume of 20 ll using random hexamers, as recommended by the manufacturer.
In some cases, total RNA was treated with 1 unit of
RQ1 RNase-free DNase (Promega, Madison, WI) for
60 min at 37 °C followed by heating for 5 min at 75 °C
prior to reverse transcription. Real-time PCR SYBR
Green I assays were performed in triplicate for each
sample using an ABI Prism 7900HT sequence detection
system (Applied Biosystems). Primers were designed
173
using Primer Express Software (Applied Biosystems) as
follows: for P450R, forward primer 50 -CATGTCCG
CAGACCCTGAA-30 , reverse primer 50 -CGTATGTG
GCCATGCAGAAG-30 (the corresponding amplicon
spans 710 nucleotides of P450R intron 4 [10], thus
making amplification of the P450R gene in contaminating genomic DNA less likely under the conditions of
real-time PCR); for 18S rRNA, forward primer 50 CGCCGCTAGAGGTGAAATTC-30 , reverse primer
50 -CCAGTCGGCATCGTTTATGG-30 . The reaction
mixture for the real-time PCR assay (total volume
4–10 ll) contained SYBR Green I PCR master mix
(Applied Biosystems), 300 nM of each primer, and 200–
1000 pg (for P450R assay) or 1.7–10 pg (for 18S rRNA
assay) of reverse transcribed liver cDNA. Relative
quantitation of P450R RNA with normalization to 18S
rRNA was performed using the standard curve method,
as described in the manufacturerÕs User Bulletin 2
(Applied Biosystems). Dissociation curves were generated after each real-time PCR run to ensure that a single
specific product was amplified during the run.
In vitro translation. In vitro translation of total RNA
or mRNA prepared from individual liver samples was
carried out using reticulocyte lysate (Ambion, Austin,
TX, or Promega). Reaction mixtures (25 ll total volume) contained commercially supplied buffer, 9–20 lg
total RNA or 0.8 lg mRNA, 20 U RNasin (Promega),
16.7 ll reticulocyte lysate, and 2.1 ll of [35 S]methionine
(Perkin Elmer Life Sciences, Boston, MA). In some experiments, 8–70 lg of liver cell extract from untreated or
T3-treated hypothyroid rats was added to the reaction
mixtures (for preparation of liver extracts, see below).
The translation reaction was carried out at 30 °C for
60–90 min and stopped by placing the tubes on ice.
Duplicate aliquots (2 ll each) were removed from
each mixture to assess the overall level of radiolabel
incorporation by precipitation of the proteins with
12.5% trichloroacetic acid (w/v). Precipitates were collected on glass fiber filters and counted in a liquid
scintillation counter. The remaining portion of each
translation mixture (21 ll) was sequentially immunoprecipitated, first with anti-P450R antibody and then
with anti-GST antibody. Immunoprecipitation was
performed in 100 mM sodium phosphate buffer, pH 7.4,
containing 0.9% NaCl, 1 mM EDTA, 0.1% Triton X100, 0.5% Nonidet P-40, 1 mg/ml BSA, and Complete
protease inhibitor cocktail (Roche Diagnostics GmbH,
Mannheim, Germany) in a total volume of 400–500 ll.
Translation reactions were incubated overnight with
rabbit polyclonal P450R antibody (final dilution 1:400)
[19], followed by removal of the anti-P450R-antigen
complex using protein A–Sepharose CL-4B (Amersham
Pharmacia Biotech, Uppsala, Sweden). The supernatants were subsequently incubated for 5 h with a rabbit
polyclonal anti-GSTa antibody (final dilution 1:500).
Immunoprecipitated proteins were separated on 8.8%
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SDS–PAGE gels, which were analyzed with a Storm
PhosphorImager and ImageQuant software (Molecular
Dynamics, Sunnyvale, CA).
Preparation of liver extracts. A cytosolic extract was
prepared from frozen livers using a modification of a
method previously described [20]. All steps were performed at 4 °C. Liver tissue (1 g) pooled from two
untreated hypothyroid rats or from two T3-treated hypothyroid rats killed 12 h after T3 treatment was homogenized in 5 ml of homogenization buffer (10 mM
Tris–HCl, pH 7.6, 1 mM EDTA, 250 mM sucrose)
containing 2 Complete protease inhibitor mixture
(Roche Diagnostics GmbH), 10 mM NaF, and 1 mM
Na3 VO4 . Homogenates were centrifuged at 9000 rpm
for 20 min in a Sorvall SA-600 rotor, followed by centrifugation of the post-nuclear supernatant for 1 h at
45,000 rpm in a Sorvall T-1270 rotor. Proteins were
precipitated by adding solid ammonium sulfate to 60%
saturation, collected by centrifugation for 2 min at
14,000 rpm in a microcentrifuge, dissolved in buffer A
(20 mM Tris–HCl, pH 7.4, 20 mM KCl, 7 mM bmercaptoethanol, 0.1 mM EDTA, and 10% glycerol),
and dialyzed overnight against buffer A. After dialysis,
the extracts were briefly centrifuged to remove any undissolved material, and aliquots were stored at )80 °C.
Northern blotting. Northern blotting of total liver
RNA samples (30 lg/lane) was performed using
1% agarose gels containing 1.2 M formaldehyde as
described earlier [14]. The gels were probed with
the following oligonucleotide probes and hybridization conditions: 50 -gCGCGGTCCTGTAGGTCTCT-30
(probe ON-762, complementary to rat P450R mRNA
untranslated exon 1, nt 1–19; probe hybridized at 45 °C
in the presence of 15% formamide) and 50 -TAGCGGCCCTTGGTCATCAG-30 (probe ON-28, complementary to rat P450R mRNA exon 16, nt 1999–2019;
probe hybridized at 50 °C in the presence of 10%
formamide) [14]. Oligonucleotide probe ON-4 complementary to rat P450 2C6 mRNA and hybridization
conditions were described previously [13].
Preparation of microsomes. Portions of individual
frozen livers (200–400 mg) were homogenized on ice in a
mechanical homogenizer (8–9 strokes) with 2 ml of
10 mM Tris–HCl buffer, pH 7.6, containing 1 mM
EDTA, 250 mM sucrose, 2 Complete protease inhibitor mixture (Roche Diagnostics GmbH), 10 mM NaF
and 1 mM Na3 VO4 . Samples were centrifuged for
20 min at 9000 rpm at 4 °C in a Sorvall SA-600 rotor.
The supernatants were then centrifuged for 1 h at 45,000
at 4 °C in a Sorvall T-1270 rotor. Microsome pellets
were resuspended in 100 mM potassium phosphate
buffer, pH 7.4, 0.1 mM EDTA, and 20% glycerol, aliquoted, and stored at )80 °C.
Enzymatic activity and western blotting. Liver microsomal P450R activity was assayed by cytochrome c reduction [21]. Western blotting was performed using
polyclonal rabbit antibody raised to rat liver P450R [19].
Primary antibody dilution was 1:3000.
Polysome preparation and fractionation. Liver polysome preparation and polysome fractionation were
performed as described [22]. Briefly, rat livers were homogenated in RS buffer (200 mM Tris–HCl, pH 8.5,
50 mM KCl, 40 mM MgCl2 , 25 mM EGTA, 0.25 M sucrose, 5 mM dithiothreitol, 1 unit/ll RNasin (Promega)
and 2 lg/ml cycloheximide). Triton X-100 was added to
the homogenates to a final concentration of 2.5%, and
the mixtures were centrifuged for 16 min at 12,000g. The
supernatants were applied on a discontinuous sucrose
gradient consisting of an upper cushion of 1.0 M sucrose
and a lower cushion of 1.9 M sucrose, both in RS buffer
containing 0.25 U/ll RNasin. The samples were centrifuged for 2.5 h at 250,000g at 4 °C. Polysomal pellets
were resuspended in 1.25 ml RS buffer and stored frozen.
To size-fractionate the polysomes, the polysomal pellets
(0.25 ml) were loaded onto 10 ml, 15–57% linear sucrose
gradients. Samples were centrifuged for 105 min at
250,000g at 4 °C. Twelve fractions were collected using a
1-ml pipet, with the polysome peak (A260 ) typically localized to fractions 7–8. Total RNA was prepared from
polysomes and polysomal fractions using TRIzol reagent (Invitrogen) and analyzed by Northern blotting as
described above.
Results
We first investigated the effects of T3 treatment on
liver P450R mRNA, protein, and activity. Euthyroid
rats were given a single, receptor-saturating dose of T3
and then killed at times ranging from 12 to 36 h later.
Total liver RNA was isolated and P450R mRNA levels
assayed by two methods: Northern blotting with an
antisense oligonucleotide probe complementary to
P450R untranslated exon 1; and real-time PCR amplification of a 100-bp P450R cDNA fragment spanning
the junction between P450R exon 4 and exon 5 (Fig.
1A). Microsomes prepared from the same livers were
assayed for P450R protein and P450R activity (Fig. 1B).
Induction of P450R mRNA was readily detected at all
time points tested (12, 16, 24, and 36 h). Real-time PCR
analysis showed that P450R mRNA was increased 3fold 24 h after T3 injection (panel A, bottom right).
Northern blot analysis gave a slightly different induction
pattern (panel A, top, and bottom left); depending on
the background correction method, the induction value
varied from 2.5- to 4-fold.
In contrast to P450R mRNA, microsomal P450R
protein and activity levels were only slightly altered by
T3 treatment (Fig. 1, panel B). P450R protein increased
30% by 16 h, while P450R activity (cytochrome c reduction) was virtually unchanged over the 36-h time
course. In a separate experiment, where euthyroid rats
E.V. Apletalina et al. / Archives of Biochemistry and Biophysics 409 (2003) 172–179
175
T3 does not alter the association of P450R mRNA with
polysomes
To investigate whether translational regulation could
potentially explain the discoordinate changes in P450R
mRNA and protein levels seen in hyperthyroid rats, we
tested whether T3-induced P450R mRNA is poorly associated with polysomes, which would result in low efficiency of P450R protein synthesis. To determine if such
a mechanism is operative, we isolated polysomal fractions from livers of untreated and T3-treated rats. Triton X-100-treated liver homogenates were centrifuged
through a discontinuous sucrose gradient, following
which total liver RNA was purified from the polysomes
and analyzed on Northern blots probed with an oligonucleotide complementary to P450R mRNA. As evident
in Fig. 2 (panel A, top), the fold-induction by T3 of
polysome-associated P450R mRNA was at least as great
as that seen for the total liver RNA fraction. Reprobing
for P450 2C6 mRNA, which is not responsive to T3 [23],
served as a loading control (Fig. 2, panel A, bottom).
Fig. 1. Effect of T3 treatment on P450R mRNA, protein and activity in
rat liver. Euthyroid male rats were given a single i.p. injection of T3
and were killed 12, 16, 24, or 36 h later (three rats per time point).
Control rats (three animals) received a vehicle injection and were killed
12 h later. Total RNA and microsomes were isolated from individual
livers. Panel A. T3 induction of P450R mRNA. Top: Northern blot
analysis of total RNA samples (30 lg) resolved on a 1% agarose/1.2 M
formaldehyde gel and probed with a single-stranded oligonucleotide
complementary to P450R mRNA exon 1 (ON-762). The large variation in P450R mRNA in control livers seen in this experiment (lanes 1–
3) is not typical. In other experiments, the variation in P450R mRNA
in control livers was considerably smaller. Bottom left: Phosphorimager quantitation of P450R mRNA Northern blot bands. Bottom
right: Quantitation of P450R mRNA by real-time PCR. Shown is the
fold-induction of P450R mRNA following T3 treatment after normalization to 18S rRNA. Real-time PCR assays of P450R mRNA and
18S rRNA were performed following reverse transcription of total
RNA samples from individual livers. The difference in fold-induction
of P450R mRNA obtained by Northern blot analysis and by real-time
PCR may be partially attributed to the difficulty of quantitating the
low intensities of Northern blot bands in control samples (lanes 1 and
2). Panel B. Effect of T3 on liver P450R protein and activity. Shown is
a Western blot of P450R in liver microsomes (45 lg) (top) with
quantitation of the band intensities (bottom, left). P450R activity
(cytochrome c reduction rates) was assayed in isolated liver microsomes as described in Materials and methods (bottom, right). Data
shown here are mean values SEM (n ¼ 3).
were killed 12 and 24 h after T3 injection, T3 stimulated
a 2.7-fold induction of P450R mRNA by 24 h, whereas
P450R protein and activity increased by only 40% and
30%, respectively (data not shown). These data support
the conclusion that the induction of P450R mRNA by
T3 is not accompanied by a correspondingly large increase in P450R protein and activity.
Fig. 2. Effect of T3 on association of P450R mRNA with polysomes.
Panel A. T3 induction of polysome-associated P450R mRNA. Euthyroid male rats (two rats per group) were given a single i.p. injection
of T3 or a vehicle and were killed 12 h later. Liver homogenates from
these rats were treated with Triton X-100 to release polysomes from
the endoplasmic reticulum membrane, and polysomes were prepared
by passing through a discontinuous 1.0 M/1.9 M sucrose gradient.
Polysomal RNA and total RNA from control rats (lanes 1, 2, 5, 6) and
T3-treated rats (lanes 3, 4, 7, 8) was analyzed by Northern blotting
with oligonucleotide probes specific to P450R (ON-28) (top) or P450
2C6 mRNA (bottom). Panel B. Polysomal size distribution of P450R
mRNA. Polysomes isolated from T3-treated (top) and control (bottom) rat liver were fractionated on a 15–57% linear sucrose gradient.
Total RNA purified from the collected fractions was analyzed by
Northern blotting with a P450R-specific oligonucleotide probe.
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This finding suggests that T3 treatment does not lead to
inefficient association of P450R mRNA with polysomes.
Further support for this conclusion was obtained by
analysis of P450R polysome size distribution when
fractionated on a linear sucrose gradient and analyzed
by Northern blotting. As shown in Fig. 2, panel B, the
polysomal size distribution of P450R mRNA was not
altered by T3 treatment. We conclude that the absence
of a large increase in P450R protein in T3-treated rat
liver does not reflect decreased recruitment of P450R
mRNA into high molecular weight polysomes.
P450R mRNA isolated from untreated and T3-treated
rats has a similar in vitro translation efficiency
T3-induced structural changes in P450R mRNA
could potentially account for the discoordinate changes
in P450R mRNA and P450R protein and activity seen in
T3-treated rats. Indeed, T3 stimulates an increase in liver P450R poly(A) tail length 12 h after hormone
treatment [14]. To determine whether T3-induced P450R
mRNA has an intrinsically poor translation efficiency
due to this or other structural alterations, total liver
RNA isolated from control rats and from 12 or 24 h T3treated euthyroid rats was translated in an in vitro
translation system in the presence of [35 S]methionine.
Translation reactions were sequentially immunoprecipitated with anti-P450R antibody and anti-GST antibody, followed by SDS–PAGE and phosphorimaging.
Fig. 3, panel A, shows that the yield of P450R protein in
an in vitro translation system increased when 12-h or 24h T3-induced total liver RNA was used as a template as
compared to uninduced total liver RNA (lanes 3–6 vs
lanes 1 and 2). When the intensities of the 35 S-labeled
P450R bands (lanes 1–6) were normalized to the intensities of the GST bands (lanes 7–12), the yield of P450R
obtained with 12-h and 24-h T3-induced RNA was, respectively, 2.2 and 2.7 times higher than that obtained
with control RNA. These values are similar to the foldinduction of P450R mRNA seen in the same RNA
samples (data not shown). Thus, P450R mRNA isolated
from all three groups of rats (untreated euthyroid rats,
and euthyroid rats killed 12 and 24 h after T3 treatment)
is translated with a similar efficiency in vitro.
To confirm our finding that T3 treatment does not
affect the translation efficiency of P450R mRNA, we
repeated the in vitro translation experiments using liver
mRNA from untreated and T3-treated hypothyroid
rats. Methimazole-induced hypothyroid rats show a response similar to that seen in euthyroid rats: a single
injection of T3 at a receptor-saturating dose substantially increases P450R mRNA with little change in
P450R protein and activity [11]. T3 induction of liver
P450R mRNA in the hypothyroid rats used for these in
vitro experiments was significantly higher (18-fold)
than in the euthyroid rats (3-fold) examined in Fig. 3,
Fig. 3. T3-induced P450R mRNA is translated in vitro with efficiency
comparable to that of uninduced P450R mRNA. Panel A. In vitro
translation of total liver RNA from T3-treated and untreated euthyroid rats. Euthyroid rats (two rats per group) received a single i.p.
injection of T3 or a vehicle and were killed 12 or 24 h later, as marked.
Equal quantities of total RNA (9 lg) were translated in a reticulocyte
in vitro translation system in the presence of [35 S]methionine, followed
by sequential immunoprecipitation with anti-P450R (lanes 1–6) and
anti-GST antiserum (lanes 7–12). The samples were analyzed by SDS–
PAGE followed by the phosphoimager analysis. Panel B. In vitro
translation of mRNA from T3-treated and untreated hypothyroid rats.
Hypothyroid male rats (two animals per group) received a single i.p.
injection of T3 or a vehicle (as marked) and were killed 12 h later.
mRNA (0.8 lg) purified from individual livers was translated in a reticulocyte in vitro translation system. The other conditions were the
same as in panel A.
panel A (data not shown), making it easier to evaluate
the impact of T3 treatment on the efficiency of P450R
mRNA translation in vitro. In vitro translation of liver
mRNA from untreated and 12 h T3-treated hypothyroid
rats resulted in a substantially higher yield of 35 S-labeled
P450R protein in the case of the T3-induced mRNA
(Fig. 3, panel B; lanes 3, 4 vs lanes 1,2; cf. GST controls
in lanes 5–8), in agreement with the much higher P450R
mRNA content of the T3-treated liver samples. We
conclude that T3 treatment does not affect the in vitro
translation efficiency of P450R mRNA, suggesting that
the poor responsiveness of P450R protein to T3 is not
due to structural changes in P450R mRNA.
E.V. Apletalina et al. / Archives of Biochemistry and Biophysics 409 (2003) 172–179
Fig. 4. Effect of liver cytoplasmic extracts from T3-treated and untreated rats on in vitro translation of P450R. Cytoplasmic extracts and
total RNA were prepared from livers of hypothyroid rats that were
given a single i.p. injection of T3 or a vehicle and were killed 12 h later.
Different amounts of the cytoplasmic protein extract (as marked) were
added to a reticulocyte in vitro translation reaction containing
[35 S]methionine and total RNA isolated from either a T3-treated or an
untreated rat (15 and 20 lg, respectively). The translation products
were immunoprecipitated sequentially with anti-P450R (top panel) and
anti-GST antiserum (bottom panel) and analyzed by SDS–PAGE
followed by phosphoimager analysis.
Liver cytosolic proteins do not alter the translation of
P450R mRNA in response to T3 treatment
Another possible mechanism for translational regulation of P450R could involve induction by T3 of a
soluble trans-acting protein that binds to P450R mRNA
and inhibits its translation. To determine if such a transacting protein is induced by T3, we prepared extracts
from livers of untreated and 12 h T3-treated hypothyroid rats. Total liver RNA was translated in vitro in the
presence of [35 S]methionine and in the presence or absence of the soluble liver extract. Immunoprecipitation
analysis was performed using anti-P450R antibody and
anti-GST antibody, as described above, followed by
SDS–PAGE and phosphorimaging. Liver cytosolic extracts from untreated and T3-treated hypothyroid rats
exhibited a nonspecific inhibition of the translation reaction, as indicated by the decrease in the overall
[35 S]methionine incorporation in the presence of either
liver extract (up to 55% decrease in the presence of 52 lg
of liver cytosolic protein; data not shown). However,
neither of the liver extracts exerted a specific inhibitory
effect on the translation of P450R mRNA (Fig. 4). By
contrast, T3-treated rat liver extract was somewhat
more inhibitory to the translation of the GST mRNAs
as compared to untreated rat liver extract (cf. lanes 2, 4,
6, 9, 11 vs lanes 1, 3, 5, 8, 10). This observation is
consistent with the report that GST is regulated by
thyroid hormone at a post-transcriptional level [24].
Overall, we did not find any trans-acting factors in the
liver extracts that affected the translation of P450R
mRNA in a T3-dependent manner.
Discussion
Normal thyroid hormone levels are required for
maintenance of P450R mRNA, protein, and activity, as
177
well as P450R-dependent P450 monooxygenase activity,
in rat liver [11,21]. T3 has been shown to regulate P450R
at the transcriptional level [13], and also post-transcriptionally, through effects on P450R mRNA stability
and poly(A) tail length [14]. Further evidence for posttranscriptional effects of T3 on P450R is provided by the
finding that T3 treatment of rats induces a substantial
increase in liver P450R mRNA with little or no changes
in P450R protein and activity levels. The present study
extends our previous observations regarding this discoordinate regulation of P450R protein/activity vs P450R
mRNA [11] and explores several possible mechanisms of
translational regulation that could underlie this response.
Translation can be regulated by several mechanisms,
including the association of mRNA with polysomes,
which provides a general indication of the efficiency of
translational initiation. A classic example of this type of
control is the regulation of ferritin mRNA translation
by iron, where the binding of iron regulatory protein to
the 50 -untranslated region (50 -UTR) of ferritin mRNA
results in the dissociation of ferritin mRNA from
polysomes [25]. T3 can modulate this binding of the iron
regulatory protein to ferritin mRNA [26]. To determine
if T3 treatment of rats resulted in an inefficient association of P450R mRNA with polysomes, we analyzed
polysomes prepared from control and T3-stimulated
euthyroid rats. The fold-induction by T3 of polysomebound P450R mRNA was found to be similar to that
seen when analyzing total liver P450R RNA. Moreover,
no shift in the polysome size distribution of P450R
mRNA was seen when comparing T3-treated vs untreated rat livers. These data indicate that T3 does not
selectively inhibit polysome association of P450R
mRNA and thus is not likely to inhibit P450R mRNA
translation initiation by such a mechanism.
T3 treatment could conceivably render P450R
mRNA poorly translatable due to a change in mRNA
structure. To examine this possibility, total liver RNA
and liver mRNA were translated in vitro in reticulocyte
lysates. We found that P450R mRNAs from T3-treated
and untreated rats were translated with similar efficiencies, indicating that the intrinsic translatability of P450R
mRNA is not altered by T3. We previously found that
T3 treatment of euthyroid rats leads to a transient increase in P450R mRNA poly(A) tail length, which is
detectable in the cytoplasm 12 h after hormone treatment and is reversed by 16 h [14]. An increase in poly(A)
tail length is often associated with an increase in mRNA
translation [27,28]. In the present study, however, no
difference was found in the efficiency of translation in
vitro between P450R mRNA from control euthyroid
rats vs. P450R mRNA from hyperthyroid rats, killed
12 h after T3 injection. These data suggest that the
presence of a longer poly(A) tail does not in and of itself
alter the translational efficiency of P450R mRNA. We
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cannot exclude the possibility, however, that certain
cytosolic liver proteins, which were absent from the in
vitro translation system used in these experiments, could
affect the translation of P450R mRNA in a manner that
depends on P450RÕs poly(A) tail length.
Translational regulation can also occur through interactions of trans-acting proteins with either the 50 - or
30 -UTR of an mRNA. T3 regulation of lipoprotein lipase in rat adipocytes involves such a mechanism [29].
The trans-acting proteins described to date act as negative regulators of translation [28,30]. The inhibition of
translation by protein–mRNA interactions does not
necessary involve changes in the association of mRNA
with polysomes. Examples include the regulation of lipoprotein lipase by epinephrine [20] and under conditions of diabetes [31]. To assay if trans-acting binding
factors are involved in the inhibition of P450R mRNA
translation in response to T3 treatment, we prepared
liver extracts from untreated and T3-treated hypothyroid rats. Total RNA from untreated or T3-treated
hypothyroid rats was translated in vitro in the presence
of either liver extract. We found no specific inhibitory
effect of cytosolic extract from T3-treated rats on P450R
mRNA translation as compared to extract from untreated animals. These data suggest that trans-acting
binding proteins are not involved in the regulation of
liver P450R mRNA in response to T3.
Thus, we assessed several potential mechanisms of
P450R translational control and did not find any of
them to be involved in the regulation of P450R by T3.
Taken together, our findings suggest that P450R protein
levels may be regulated by T3 post-translationally. The
discoordinate changes in P450R mRNA vs protein and
activity levels seen in T3-treated liver may reflect, at
least in part, an increase in the rate of P450R protein
degradation. Thyroid control of gene expression at the
level of protein degradation has been shown for type I
collagen [32]. In cultured rat hepatic Ito cells, T3 treatment decreases type I collagen production by 3-fold by
decreasing the intracellular half-life of collagen [32].
Determination of the effects of T3 on the degradation of
P450R protein in vitro or, preferably, on the half-life of
P450R protein in liver in vivo or in primary hepatocyte
cultures should help determine if the regulation of
P450R by T3 involves the control of P450R protein
degradation.
It is uncertain whether the overall pattern of P450RÕs
response to T3, i.e., substantial induction of P450R
mRNA with little change in P450R protein, is limited to
T3 treatment, or whether it occurs with other inducers
of P450R as well. Treatment of rats with the classical
cytochrome P450-inducing agents phenobarbital and
dexamethasone results in a significant increase in cytoplasmic P450R mRNA, 10-fold for phenobarbital and
7-fold for dexamethasone within, respectively, 6 h and
9 h of inducer treatment [33,34]. Unfortunately, P450R
protein and activity levels were not measured at the time
points when the maximal increases in P450R mRNA
were observed. However, the increase in P450R protein
and activity in phenobarbital-treated rat liver may be
substantially lower than the increase in P450R mRNA,
as suggested by our finding that daily phenobarbital
treatment of rats for 4 days induces liver P450R mRNA
3.4-fold, whereas liver microsomal P450R activity increases by only 70% (E.V.A. and D.J.W, unpublished
data). Thus, the discoordinate regulation of P450R
mRNA and protein/activity seen in hyperthyroid rat liver may reflect a more general requirement for hepatocytes to maintain the level of P450R activity within a
relatively narrow (2–3-fold) range and to avoid strong
P450R overexpression, which may be associated with
reactive oxygen toxicity. Further investigation is required to address this point and to fully elucidate the
multiple responses of P450R to changes in hormonal
and environmental factors that regulate P450R synthesis
and degradation.
Acknowledgments
This article is dedicated to Dr. Ronald W. Estabrook
in appreciation of his outstanding scientific leadership,
as well as his mentorship and support for the past 20
years. Supported in part by National Institute of Health
Grant DK53767 (to D.J.W.).
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